We have developed a general method for the enrichment and identification of sequence specific DNA binding proteins. A well-characterized protein-DNA interaction is used to isolate proteins from crude cellular extracts or fractions thereof, which bind to specific DNA sequences based solely on this property. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. Upon addition of IPTG, the lac repressor releases the bound DNA and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method by using the lambda repressor, another well characterized DNA binding protein as a model. In addition, using crude preparations of the yeast mitochondrial RNA polymerase, we have identified a 70,000 MW peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.